Investigating the mechanism of TNFalpha mediated cell death in oligodendrocytes

该方案的目的是探索少突胶质细胞(OG)偏爱的分子机制 坏死性下垂作为主要的细胞死亡机制及其作用机制 在动物模型中导致进行性脱髓鞘、炎症和神经变性的这一途径 多发性硬化(MS)。MS是一种中枢神经系统(CNS)的炎症性脱髓鞘疾病， 年轻人在最具生产力的年龄最常见的慢性神经退行性疾病。 虽然多发性硬化症的免疫学基础已经被广泛研究，但我们对多发性硬化症的免疫学基础仍然知之甚少。 导致OGs退变的机制，OGs是产生髓鞘的细胞，在 维持神经元轴突的活性和完整性。预防OGs的死亡可能能够抑制 脱髓鞘和轴突变性是慢性阻塞性肺疾病患者不可逆神经功能障碍的主要原因 进展性多发性硬化 最近研究表明，由肿瘤坏死因子激活的TNFR1介导了两种不同的细胞死亡途径： Caspase依赖的细胞凋亡和caspase非依赖的RIP1激酶依赖的坏死性下垂(程序性 坏死)。然而，对于到目前为止分析的大多数细胞类型，坏死性下垂只有在激活了 半胱氨酸氨基转移酶被化学抑制剂或基因突变所抑制。有趣的是，我们发现OG经历了 肿瘤坏死因子单独刺激时的坏死性下垂，可被NEC-1或RIP3缺乏症有效阻断。 我们已经证明，7-Cl-O-Necrostatin-1(7-Cl-O-NEC-1)，一种高度特异的RIP1激酶抑制剂，可以保护 抗肿瘤坏死因子诱导的少突胶质细胞死亡和两种体内多发性硬化症小鼠模型 和实验性自身免疫性脑脊髓炎模型(EAE)。此外，RIP3-/-小鼠也对 铜原酮模型和RIP3-/-OGs可免受肿瘤坏死因子的影响。我们建议调查一下为什么OG 倾向于将坏死性下垂作为主要的细胞死亡途径以及RIP1激酶的作用和机制 调停奥格斯之死。具体目标1是研究细胞的作用和机制 代谢和氧化还原状态控制OGs对肿瘤坏死因子的敏感性。这是为了测试细胞的假设-- 细胞相互作用调节OGs的高代谢活性提供了控制氧化还原状态的关键机制 OGs对肿瘤坏死因子介导的坏死性下垂的敏感性。具体目标二是调查S的角色-- 亚硝酸化在调节OGs对肿瘤坏死因子诱导的细胞死亡敏感性中的作用这是为了检验这一假设 肿瘤坏死因子刺激的OGs亚硝化应激导致半胱氨酸天冬氨酸氨基转移酶抑制 从OGS到坏死性下垂。特异性目标3是研究RIP1激酶在介导坏死性上睑下垂中的作用。 OGS在体内和体外使用RIP1激酶死亡敲打突变小鼠。我们的研究可能会提供一个强有力的 开发RIP1激酶抑制剂作为治疗多发性硬化的OG保护性策略的理论基础， 以及作为先导化合物的口服可用、高度特异和无毒的RIP1激酶抑制剂7-Cl-O-NEC-1。

The goal of this proposal is to explore the molecular mechanism as to why oligodendrocytes (OGs) prefer necroptosis, a regulated necrotic cell death pathway, as the primary cell death mechanism and the contribution of this pathway to progressive demyelination, inflammation and neurodegeneration in animal models of multiple sclerosis (MS). MS, an inflammatory demyelinating disease of the central nervous system (CNS), is the most common chronic neurodegenerative disease for young adults during their most productive ages. While the immunological basis of MS has been studied extensively, we still know very little about the mechanism that leads to the degeneration of OGs, the myelin producing cells that play a critical role in the maintenance of activity and integrity of neuronal axons. Preventing the death of OGs might be able to inhibit demyelination and axonal degeneration, the major cause of irreversible neurological disability in patients with progressive MS. Activation of TNFR1 by TNF¿ has recently been shown to mediate two alternative cell death pathways: caspase-dependent apoptosis and caspase-independent RIP1 kinase-dependent necroptosis (programmed necrosis). However, for most cell types analyzed so far, necroptosis is only activated when the activation of caspases is inhibited by chemical inhibitors or by genetic mutation. Interestingly, we found that OGs undergo necroptosis upon stimulation by TNF¿ alone which can be effectively blocked by Nec-1 or by RIP3 deficiency. We have shown that 7-Cl-O-necrostatin-1 (7-Cl-O-Nec-1), a highly specific inhibitor of RIP1 kinase, protects against TNF¿-induced oligodendrocyte death in vitro and two mouse models of MS in vivo [cuprizone model and experimental autoimmune encephalomyelitis model (EAE)]. In addition, RIP3-/- mice are also resistant to cuprizone model and RIP3-/- OGs are protected against TNF¿. We propose to investigate as to why OGs prefer to use necroptosis as the primary cell death pathway and the role and mechanism of RIP1 kinase in mediating the death of OGs. Specific Aim 1 is to investigate the role and mechanism by which cellular metabolism and redox state control the sensitivity of OGs to TNF¿. This is to test the hypothesis that the cell- cell interaction regulated high metabolic activity in OGs provides a critical mechanism that controls redox state and the sensitivity of OGs to TNF¿ mediated necroptosis. Specific Aim 2 is to investigate the role of S- nitrosylation in regulating the sensitivity of OGs to TNF¿ induced cell death. This is to test the hypothesis that elevated nitrosylation stress in TNF¿ stimulated OGs leads to the inhibition of caspases and sensitization of OGs to necroptosis. Specific Aim 3 is to examine the involvement of RIP1 kinase in mediating necroptosis of OGs in vivo and in vitro using RIP1 kinase dead knockin mutant mice. Our study may provide a strong rationale for the development of RIP1 kinase inhibitors as an OG protective strategy for the treatment of MS, and an orally available, highly specific and nontoxic RIP1 kinase inhibitor, 7-Cl-O-Nec-1, as a lead compound.